The present invention relates to predator bacteria that have biocontrol activity against microorganisms. More particularly, the present invention is directed to a novel, non-obligate predator bacterium Burkholderia casidae (including variants thereof). The invention is also directed to methods for isolating and producing substantially pure cultures of Burkholderia casidae, and to antimicrobial preparations produced from such cultures. The invention is further directed to biocontrol compositions comprising such pure cultures, extracts and filtrates of such cultures, cell fractions prepared from such cultures, and antimicrobial preparations produced from Burkholderia casidae. The invention is additionally directed to methods for protecting plants against microbial diseases by treatment with biocontrol compositions of the invention.
Past attempts to control plant microbial diseases have included the use of chemicals. However, many chemicals that have been in long-time use are now ineffective due to the increasing number of chemical-resistant strains of plant pathogens. Further, many such chemicals are recognized to be potentially hazardous to non-target organisms, particularly humans, and to the environment.
Biological control of plant pathogens is an alternative to chemical control. It has been recognized that crops grown in some soils naturally are resistant to certain fungal pathogens. Furthermore, soils that are conducive to the development of fungal diseases can be rendered suppressive, or resistant, by the addition of small quantities of soil from a suppressive field. Scher et al., Phytopathology 70:421 (1980). Conversely, suppressive soils can be made conducive to fungal diseases by autoclaving or chemical fumigation, indicating that the factors responsible for disease control are biological. Root colonizing bacteria have been shown to be responsible for this phenomenon, which is known as biological disease control. Baker et al., Biological control of plant pathogens, Freeman Press, San Francisco (1974).
In many cases, the most efficient strains of biological disease controlling bacteria are fluorescent Pseudomonads. Weller et al., Phytopathology, 73: 463-469 (1983). Many biological control Pseudomonas strains produce metabolites, such as antibiotics and sideophores, that inhibit the growth of fungal pathogens. Howell et al., Phytopathology 69: 480-482 (1979); Howell et al., Phytopathology 70: 712-715 (1980).
An important factor in biological control is the ability of an organism to compete in a given environment. Thus, it is desirable to obtain novel biocontrol agents which effectively control (e.g., retard, restraint, kill, lyse) the growth of plant pathogens, particularly fungi, and which are able to aggressively compete with indigenous bacteria and other microflora that can exist on the surfaces or the rhizosphere of the plant.
The present invention generally relates to predator bacteria. More particularly, the invention is directed to a novel, non-obligate predator bacterium Burkholderia casidae, which has biocontrol activity against a broad range of microorganisms, particularly microbial pathogens. One aspect of the invention is directed to substantially pure cultures of B. casidae (including variants), and to methods for isolating and producing such substantially pure cultures. In a preferred embodiment, the invention provides a substantially pure culture of B. casidae strain 2.2N (ATCC accession no. 55961).
Another aspect of the invention is directed to preparations of antimicrobial compounds produced by B. casidae, and to methods for producing such antimicrobial preparations. In a preferred embodiment, the invention provides antimicrobial preparations comprising antifungal and anti-yeast compounds produced by B. casidae strain 2.2N or variants thereof, and methods for producing such preparations.
An additional aspect of the invention is directed to biocontrol compositions comprising materials obtained or derived from B. casidae, cells or cultures, and to methods for producing such biocontrol compositions. In one embodiment, the biocontrol compositions comprise live B. casidae cells or cultures. In another embodiment, the biocontrol compositions comprise inactivated B. casidae cells or cultures that may have been processed in a variety of ways including cell breakage and spray drying. In an additional embodiment, the biocontrol compositions comprise cell-free filtrates or cell fractions prepared from B. casidae cells or cultures. In a further embodiment, the biocontrol compositions comprise preparations of antimicrobial compounds isolated from B. casidae, cells or cultures, or cell-free filtrates or cell fractions prepared from such cultures or cells. In preferred embodiments, the biocontrol compositions comprise cells (living or dead) or cultures (living or dead) of B. casidae strain 2.2N, or cell-free filtrates or cell fractions, or antimicrobial, particularly anti-fungal, preparations made from such cells or cultures.
A further aspect of the invention is directed to methods for the prevention and/or treatment of plant microbial diseases using biocontrol compositions of the invention. In one embodiment, the methods for prevention and/or treatment comprise treating the plants themselves with an effective amount of a biocontrol composition. In another embodiment, the methods for prevention and/or treatment comprise treating plant seeds prior to planting with an effective amount of a biocontrol composition. In a further embodiment, the methods for prevention and/or treatment comprise treating the soil in the immediate vicinity of the plant with an effective amount of a biocontrol composition. In preferred embodiments, the methods of the invention are directed to prevention and/or treatment of plant fungal diseases using biocontrol compositions of the invention.
B. casidae (including variants thereof) of the invention exerts biocontrol activity against a wide spectrum of microorganisms. B. casidae adversely affects the growth or survival of such microorganisms in its vicinity. Because some of the antimicrobial activity of B. casidae is present in cell-free culture-filtrates, biocontrol activity is soluble in water and can exert its activity against microorganisms in aqueous suspension. Antimicrobial activity does not require living cells or cell contact. Susceptible microorganisms are referred to herein as prey or targets of B. casidae. Prey of B. casidae include, but are not limited to, fungi (including yeast), algae, protozoa, and bacteria (including blue green algae) other than B. casidae. 
B. casidae exerts biocontrol activity against a particular prey microorganism in its vicinity by releasing one or more antimicrobial compounds that adversely affect the growth or survival of the prey, and/or by physically attacking the prey through attachment, subsequent parasitism and eventual destruction of the prey. Different prey microorganisms are susceptible to different combinations of such actions by B. casidae. Some prey are susceptible only to physical attack by B. casidae. Other prey are susceptible only to a particular one, or combination, of the antimicrobial compounds released by B. casidae. Yet other prey are susceptible to both physical attack and one or more of the released antimicrobial compounds.
Since many prey microorganisms of B. casidae are pathogens, the antimicrobial preparations and biocontrol compositions of the invention beneficially may be used in the prevention and/or treatment of microbial diseases of plants and animals including humans. In particular, the antimicrobial preparations of the invention may be used to manufacture medicaments for use in treatment of microbial diseases of animals, fish, and humans, and to produce bactericides and fungicides for use in controlling microbial diseases, particularly those of plants. The biocontrol compositions of the invention may be directly used to prevent and/or treat microbial, particularly fungal, diseases of plants. The antimicrobial compounds and biocontrol compositions of the invention also may be used to treat, or prevent, protozoan diseases, and to control protozoan and algal growth in aquatic environments.
The present invention may be understood more fully by reference to the following detailed description of the invention, examples of specific embodiments of the invention and the appended figure.
3.1. Definitions
In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to a term, the following definitions are given to various terms and abbreviations used herein.